Parathyroid radioimmunoassay

ABSTRACT

A method for the radioimmunoassay of parathyroid hormone in mammalian serum. The method is an improvement on previous double antibody radioimmunoassays in that spurious assay results caused by the nonspecific interaction of serum proteins with the labeled peptide is eliminated by the labeling of a specific portion of either human or bovine parathyroid molecule. The 65-84 portion of human or bovine parathyroid hormone, as radioactively labeled, is incorporated in the assay as the labeled peptide. A chicken antibody with a high affinity for the 65-84 portion of human parathyroid hormone is incorporated in the assay as the first antibody. The invention further pertains to compounds useful in practicing the method, namely X 64  -hPTH 65-84  and Y-X 64  -hPTH 65-84  wherein X is either histidyl, tyramyl, histamyl, or tyrosyl, and wherein Y is either  125  I or  131  I.

This is a division, of application Ser. No. 6/169492, filed July 15,1980, now U.S. Pat. No. 4,341,755 issued July 27, 1982.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radioimmunoassay measurement of theconcentration of mammalian parathyroid hormone and certain fragmentsthereof in biological or other fluids.

2. Description of the Prior Art

Human parathyroid hormone (i.e. intact hPTH) is an 84 immuno acidsequence with a major role in maintaining the constancy (homeostasis) ofcalcium in cells and extracellular fluid, despite marked variations incalcium intake and excretion. Parathyroid peptides (both intact hPTH andcertain hPTH fragments) are secreted from the parathyroid glands inresponse to a lowering of serum calcium; and, in the absence ofparathyroid peptide secreting tumors, high levels of serum calciuminhibit secretion by the glands. Measuring parathyroid peptides in humanserum has proven clinically to be an extremely helpful and efficienttool in the differential diagnosis and management of hypercalcemia; mostnotably, radioimmunoassay (i.e. RIA) can help to diagnose tumors andhyperplasia of the parathyroid glands. RIA detection of high bloodlevels of PTH peptides can establish that excess PTH secretion iscausing hypercalcemia. RIA's can also be useful in localizinghyperfunctioning parathyroid tissue by assaying samples obtained viavenous catheterization. PTH RIA is also useful for the diagnosis andmanagement of hypocalcemia. With rare exceptions, hypocalcemic personswith deficient PTH (hypoparathyroidism) usually have subnormal bloodconcentrations of PTH peptides. Measurement of the concentration of PTHpeptides in blood serum is frequently used to assess the status of renalosteodystrophy in renal failure patients on chronic dialysis. Serum PTHlevels are often markedly elevated in patients with renal failure due tochronic negative calcium balance, hypocalcemia, and consequent secondaryhyperparathyroidism.

In clinical parathyroid literature, a set of nomenclature has becomecommon. N-terminal hPTH is the 1-34 amino acid sequence of hPTH.Antisera having a substantially high affinity for this sequence arereferred to as N-terminal antibodies. Such antibodies will generallyalso have a high affinity for any sequence of amino acids incorporatingthe 1-34 amino acid sequence. Thus, such antibodies will have a highaffinity for intact hPTH (i.e. hPTH¹⁻⁸⁴) as well as N-terminal hPTH.C-terminal hPTH is the 35-84 amino acid sequence. Antisera having asubstantially high affinity for the 35-84 sequence are referred to asC-terminal antisera. It is now well established that only small amountsof intact hPTH and even less free N-terminal hPTH are present inperipheral human serum. The main circulating hPTH peptide is C-terminalhPTH. Accordingly, RIA's are generally directed at C-terminal hPTH wherethe purpose is diagnosing hyperthyroidism and the other maladies recitedabove.

Prior art RIA's which measure the concentration of C-terminal hPTH inhuman serum are common; however, very few are available commercially.The commercially available assays generally involve the double antibodymethod. In brief, C-terminal hPTH (and lesser quantities of intact hPTH)present in human serum compete with a radioactively labeled peptide(generally radioactively labeled beef PTH¹⁻⁸⁴) for a particular firstantibody, thereby inhibiting the binding of the first antibody to theradioactively labeled peptide. As a result of the competitiveinhibition, the ratio of first antibody bound labeled peptide to freelabeled peptide diminishes as the concentration of C-terminal hPTH (andintact hPTH) increases. As a convenient method of circumvent thedifficult task of separating bound labeled peptide from free labeledpeptide, a second antibody is added that has a substantially highaffinity for the first antibody and which forms a precipitate when boundto the first antibody bound labeled peptide. The quantity of secondantibody added is generally only sufficient to bind a portion of firstantibody bound radioactively labeled peptide. The ratio of precipitatedradioactively labeled peptide to non-precipitated radioactively labeledpeptide similarly diminishes as the concentration of C-terminal hPTH(and intact hPTH) increases. The concentration of C-terminal hPTH (andintact hPTH) in an unknown sample is obtained by comparing theinhibition observed with the inhibition produced by known amounts ofC-terminal hPTH (or intact hPTH), as presented in a standard curve. Theconcentration of C-terminal hPTH is generally represented as hPTH¹⁻⁸⁴equivalents. That is, the concentration in nanograms/ml (ng/ml) of serumis reported as if C-terminal hPTH were intact hPTH.

Most prior art double antibody PTH RIA's face a common difficulty inmeasuring the concentration of C-terminal hPTH: they often give aspurious value because of nonspecific interaction (NSI) of the labeledPTH peptide with serum proteins. That is, the labeled peptide is thoughtto react with various proteins present in the serum sample with theresult that the first antibody, which ordinarily has a high affinity forthe labeled PTH peptide, has a low or no affinity for the product of thereaction of the labeled PTH peptide with serum proteins.

In some prior art double antibody PTH RIA's, the standardized reagentsused to prepare the standard curve do not contain human serum (IsotexDiagnostics, Friendswood, Tex. 77546). In this case, standards aregenerally prepared by dissolving appropriate quantities of PTH peptides,whether human, beef, or of other sources, in an appropriate non-serumbuffer. The NSI for the unknown sample of serum will depress the valueof the ratio of precipitated to non-precipitated labeled PTH peptide,while there will be no similar depression for the standard reagents.This is so because the latter do not contain human serum and thereforethe serum proteins which cause NSI are absent. Thus, the assay willindicate a concentration that is higher than the actual concentration.The error injected into assay results by NSI can be sufficiently great,on the order of several ng's/ml of hPTH¹⁻⁸⁴ equivalents, to cause amischaracterization of an unknown sample. That is, an assay of serumtaken from a person with normal parathyroid glands could indicatehyperparathyroid disease.

Some prior art double antibody PTH RIA's seek to eliminate the effect ofNSI on assay results. One prior art method (Arnaud, C. D., et al., 50 J.Clin. Invest. 21 (1971)) incorporates serum from hypoparathyroidpatients or pooled serum stripped of PTH peptides in the standardreagents used to prepare the standard curve. This attempt to adjust forthe effects of NSI on assay results is not wholly satisfactory. Theserum added to the standard reagents is not the unknown serum to betested. The concentration of serum peptides which give rise to NSI mayvary significantly from person to person. Thus, depending upon whetherthe NSI for the unknown sample is relatively greater or less than theNSI for the serum incorporated in the standard reagents, the assay willindicate respectively concentrations lower or higher than the actualconcentration. The error injected into this type of prior art assay canalso be sufficiently great ot mischaracterize an unknown sample.

Another PTH assay approach seeks to eliminate the effects of NSI onassay results by performing the RIA on significantly diluted serum.Diluting the serum in this manner does reduce NSI. However, whilereducing the effect of NSI on assay results, diluting the serumgenerally reduces the sensitivity of the RIA to the degree that the RIAcannot detect the concentrations of C-terminal hPTH necessary todistinguish normal from abnormal functioning parathyroids.

At least one prior double antibody PTH RIA has adjusted for the effectsof NSI while retaining the necessary sensitivity of the assay (PTH II,Immuno Nuclear Corp., Stillwater, Minn. 55082). In this RIA, an unknownsample of serum is divided into two portions. Intact PTH and C-terminalhPTH are removed from one of the two portions, leaving the serumproteins behind. Removing the intact hPTH and C-terminal hPTH in thismanner is accomplished by binding the first antibody to a gel or resinto create a specific solid phase absorbent for intact PTH and C-terminalhPTH. However, removing the intact hPTH and C-terminal hPTH in thismanner requires a full cycle of incubation of the portion of the serumto be stripped. Following this cycle of incubation, both the untouchedportion of serum and the stripped portion of serum are assayed accordingto standard RIA methods. The stripped serum serves as a zero referencevalue to be subtracted from the result obtained with the untouched serumto yield results adjusted for NSI. While this method of stripping aportion of the very serum to be assayed compensates for NSI, it is notwholly satisfactory because it involves an extra cycle of incubationwhich increases the total time necessary to reach a final assay resultafter drawing a sample from a patient.

Double antibody PTH RIA's directed at human serum are primarily usefulfor diagnostic purposes and secondarily useful for research purposes.Similar double antibody PTH RIA's directed at the sera of mammals suchas rats, oxen, or cows are particularly useful for advanced research onthe functioning of parathyroids, because rats, oxen, and cows can becontrolled and manipulated in ways inappropriate for human subjects. Itis commonly known that most regions of bovine PTH and hPTH arestructurally similar, and that fragments of bovine PTH are present inbovine parathyroid glands and circulate in the blood. Furthermore, it isnow known that some regions of rat PTH and hPTh are structurallysimilar, and that fragments of rat PTH are present in rat parathyroidglands and circulate in rat blood. Because of these structuralsimilarities, some antibodies have a substantially high affinity forbovine PTH, rat PTH, and hPTH. Thus, double antibody PTH RIA'sincorporating such antibodies will measure the concentration of bovinePTH and rat PTH as well as hPTH. The effect of NSI on assay results incommon to such RIA's whether directed to bovine serum, rat serum, orhuman serum. It is thought that NSI will similarly effect assays of anymammalian serum. Thus, the various attempts in prior art double antibodyPTH RIA's to adjust for the effects of NSI are not wholly satisfactorywhen such RIA's are directed to mammalian serum.

SUMMARY OF THE INVENTION

The invention is based on the discovery that NSI can be eliminated indouble antibody PTH RIA's directed to mammalian serum, especiallybovine, rat, or human serum, by radioactively labeling only a selectedfragment of the hPTH sequence or the bovine PTH sequence. Aradioactively labeled fragment within the range of about 65-84 of bovinePTH or hPTH is incorporated in the assay as the labeled peptide.Accordingly, the invention includes a method for double antibodyradioimmunoassay measurement of the concentration of PTH in biologicalfluids which comprises incorporating as the radioactively labeledpeptide a radioactively labeled fragment within the range of about 65-84of bovine PTH or hPTH, and compounds useful in practicing the method.The compounds include those having the formula X⁶⁴ -hPTH⁶⁵⁻⁸⁴ wherein Xis a member of the group consisting of histidyl, tyramyl, histamyl, andtyrosyl and those having the formula Y-X⁶⁴ -hPTH⁶⁵⁻⁸⁴ wherein Y is amember of the group consisting of ¹³¹ I and ¹²⁵ I and wherein X is amember of the group consisting of histidyl, tyramyl, histamyl, andtyrosyl.

Double antibody PTH RIA's incorporating radioactively labeled fragmentswithin the range of about 65-84 of bovine PTH or hPTH follow methodsotherwise common to double antibody RIA's. A first antibody must befound that has a high affinity for the particular radioactively labeledfragment and the PTH peptides to be measured. The unknown sample, thefirst antibody, and the radioactively labeled fragment are addedtogether, or the addition of the radioactively labeled fragment isdelayed for enhanced sensitivity of the assay. A second antibody isadded which binds a portion of first antibody bound radioactivelylabeled fragment, thereby forming a precipitate. Precipitation may beaccelerated according to conventional means with polyethylene glycol(PEG) and pre-precipitation with normal serum.

Radioactively labeled hPTH⁶⁵⁻⁸⁴ is an example of a radioactively labeledfragment within the range of about 65-84 of hPTH. The fragment hPTH⁶⁵⁻⁸⁴may be radioactively labeled in many different ways, including adding agroup that is receptive to known radioisotopes. Accordingly, hPTH⁶⁵⁻⁸⁴can be synthesized by solid state peptide chemistry, with, for example,a histidyl, tyramyl, histamyl, or tyrosyl (Tyr) group added at the 64thposition of the hPTH sequence. The latter groups are receptive toradioiodine and other isotopes, which may be attached to the receptorgroups according to conventional means. Thus, ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ isan example of a radioactively labeled hPTH⁶⁵⁻⁸⁴ and consequently is anexample of a radioactively labeled fragment within the range of about65-84 of hPTH.

The elimination of NSI for an RIA of the present invention isillustrated by comparing its assay of mammalian serum with that of aprior art RIA (PTH II, Immuno Nuclear Corp., Stillwater, Minn. 55082).The RIA of the present invention utilizes ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ as theradioactively labeled peptide and an antibody, raised in chickensagainst intact bovine PTH, having a high affinity (1+10¹⁰ l/mol.) forhPTH⁶⁵⁻⁸⁴. This first antibody consequently also has a high affinity for¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴, intact hPTH, and C-terminal hPTH. The prior artRIA utilizes radioiodinated intact bovine PTH and an antibody, raised inchickens against intact bovine PTH, having a high affinity forhPTH⁴⁴⁻⁶⁴. This first antibody consequently also has a high affinity forradioiodinated intact bovine PTH, intact hPTH, and C-terminal hPTH.

To compare the RIA of the present invention with the prior art RIA,uremic human serum (with elevated C-terminal hPTH) was separated by asuperfine G-75 Sephadex chromatograph. The contents of each eluate tubewere divided into two portions to be assayed respectively by the twoRIA's. In the area of tubes 20-25 (i.e. the void area containing noC-terminal hPTH), the prior art RIA showed approximately 4 ng/mlhPTH¹⁻⁸⁴ equivalents (a spurious result attributable to NSI), while theRIA of the present invention showed no hPTH¹⁻⁸⁴ equivalents (hence, noNSI).

The RIA of the present invention, incorporating ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴,will assay rat serum and bovine serum as well as human serum. This is sobecause its first antibody has a substantially high affinity for intactrat PTH and certain rat PTH fragments, and because the first antibodyalso has a substantially high affinity for intact bovine PTH and certainbovine PTH fragments. Serum proteins in rat, bovine, and human serum arethought to interact nonspecifically on the same region of the hPTHmolecule when that molecule is radioactively labeled. Therefore, the RIAof the present invention by incorporating radioactively labeledhPTH⁶⁵⁻⁸⁴, whether directed at rat, bovine, or human serum, excludesthat region of the hPTH molecule which interacts with serum proteins.Thus, it does not suffer the adverse effects of NSI when directed tosuch sera.

NSI is similarly eliminated by incorporating as the radioactivelylabeled peptide radioactively labeled fragments within the range ofabout 65-84 of bovine PTH or hPTH. Serum proteins are thought tointeract nonspecifically on a region of labeled hPTH molecules andlabeled bovine PTH molecules at least somewhat removed from the regionof 65-84. This is evidenced by the above comparison of the RIA of thepresent invention with the prior art RIA. NSI in the prior art RIAoccurred somewhere in the region of 44-64. The antibody therein had anaffinity for hPTH⁴⁴⁻⁶⁴ and NSI suppressed binding of this antibody tolabeled intact beef PTH. Accordingly, radioactively labeled peptidesbased on fragments smaller than hPTH⁶⁵⁻⁸⁴ or bovine PTH⁶⁵⁻⁸⁴ in suchRIA's would also eliminate NSI. Moreover, fragments somewhat larger thanhPTH⁶⁵⁻⁸⁴ or bovine PTH⁶⁵⁻⁸⁴ could serve as the basis for theradioactively labeled peptide. For example, radioactively labeledhPTH⁶³⁻⁸⁴ or radioactively labeled bovine PTH⁶³⁻⁸⁴ would also eliminateNSI in such RIA's. Based on general immunological principles, the firstantibody of the RIA of the present invention will have a high affinityfor fragments larger than hPTH⁶⁵⁻⁸⁴ or bovine PTH⁶⁵⁻⁸⁴ and may well havea high affinity for smaller fragments. Other antibodies could be raisedwith the requisite affinities should this particular first antibody nothave a high affinity for certain smaller fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that spurious assay results due to NSI can result inmischaracterizing normal human serum as being indicative ofhyperparathyroidism.

FIG. 2 illustrates that the RIA of the present invention eliminates NSIby comparing the results of this RIA with a prior art RIA where theassays are directed to the void volume of chromatographically separatedserum.

FIG. 3 is a standard curve plotting % binding vs. concentration.

FIG. 4 is a comparison of standard curves for 50, 100, and 200 μl ofstandard and unknown serum.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following is a description of a typical preparation and operation ofa double antibody PTH RIA which incorporates radioactively labeledhPTH⁶⁵⁻⁸⁴. This RIA will assay rat, bovine, or human serum. It will alsoassay biological or other fluids.

PREPARATION OF STANDARD REAGENTS

0.1 M borate buffer (4.0 Liters)

24.75 gm boric acid

8.10 gm NaOH

9.0 ml concentrated HCl

Dissolve and dilute to 4.0 liters with distilled water. pH 8.4.

0.25 M borate buffer (1.6 Liters)

24.75 gm boric acid

8.10 gm NaOH

9.0 ml concentrated HCl

Dissolve and dilute to 1.6 liters with distilled water. pH 8.4.

5% BSA-0.25 M borate buffer (4.0 Liters)

61.88 gm boric acid

20.25 gm NaOH

22.5 ml concentrated HCl

665 ml 30% by volume BSA in distilled water (Bovine Serum Albuminavailable commerically from Sigma Chemical Co., St. Louis, Missouri63178)

8.0 ml 1% by volume merthiolate (thimersal) in distilled water.

Dissolve and dilute to 4.0 liters with distilled water. pH 8.4.

5% BSA-0.25 M borate buffer in 0.22 M EDTA (100 ml)

16.5 ml 30% BSA

8.18 gm disodium EDTA (disodium ethylenediamine tetracetic aciddihydrate)

0.2 ml 1% merthiolate

Dissolve and dilute to volume of 100 ml with 0.25 M borate buffer;adjust pH to 8.4 with 40% by volume NaOH in distilled water.

1% BSA-0.1 M borate buffer (4.0 Liters)

24.75 gm boric acid

8.10 gm NaOh

9.0 ml concentrated HCl

133 ml 30% BSA

1.6 ml 1% merthiolate

Dissolve and dilute to 4.0 liters with distilled water. pH 8.4.

0.2 M acetic acid-ammonium acetate (500 ml)

18 ml 1.0 M ammonium acetate

82 ml 1.0 M acetic acid

400 ml distilled water pH 4.0.

0.5 M Phosphate buffers (950 ml)

(a) 68.1 g/liter KH₂ PO₄

(b) 89.0 g/liter Na₂ HPO₄.2H₂ O (or 71.0 g/liter anhydrous Na₂ HPO₄)

Mix 140 ml of (a) and 810 ml of (b) to obtain phosphate buffer of pH7.4. Store at 2°-8° C. At those temperatures, the salts willprecipitate; before use, warm solution until salts redissolve. Testsolution for mold contamination by retaining sample of newly createdsolution at room temperature in an enclosed glass container. If mold ispresent, discard refrigerated solution.

0.05 M Phosphate buffers (1.0 Liter)

Dilute 100 ml 0.5 M phosphate buffer to 1.0 liter with distilled water.pH 7.4.

15% PEG-0.001% Triton (1.0 Liter)

150 grams polyethyleneglycol

0.01 gram Triton

Dissolve and dilute to 1.0 liter with distilled water.

Additional reagents necessary to the preferred embodiment may be basedon the foregoing reagents.

PREPARATION OF CHICKEN ANTI-hPTH⁶⁵⁻⁸⁴ REAGENT

Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent is the result of raising the chickenanti-hPTH⁶⁵⁻⁸⁴ antibody against intact bovine parathyroid hormone. Thisantibody was chosen for its high affinity for intact hPTH, C-terminalhPTH fragments, and radioactively labeled hPTH⁶⁵⁻⁸⁴. As alternativeembodiments, other antibodies with a substantially high affinity forthese three substances, could be utilized in a C-terminal PTH RIA kit.Such antibodies may be raised in animals other than chickens. Also suchantibodies may be raised in chickens or other animals against substancesother than intact bovine PTH. Such antibodies may be raised, forexample, against intact hPTH and certain fragments of the foregoing.

The chicken anti-hPTH⁶⁵⁻⁸⁴ antibody also has a high affinity for intactbovine PTH and bovine PTH⁶⁵⁻⁸⁴. C-terminal fragments of bovine PTH atleast as large as those fragments of bovine PTH within the range ofabout 65-84 are designated herein as C-terminal bovine PTH fragments.This antibody has a high affinity for such fragments. Furthermore, thisantibody has a high affinity for intact rat PTH. This suggests thatcertain of the larger C-terminal fragments of rat PTH are sufficientlysimilar in structure to hPTH⁶⁵⁻⁸⁴ that the antibody will have a highaffinity for such fragments. C-terminal fragments of rat PTH at least aslarge as those fragments of rat PTH within the range of about 65-84 aredesignated herein as C-terminal rat PTH fragments. It is thought thateither the chicken anti-hPTH⁶⁵⁻⁸⁴ antibody or a substitute antibody witha high affinity for radioactively labeled fragments within the range ofabout 65-84 of bovine PTH or hPTH will have a high affinity for theparathyroid hormone of any mammal. Fragments of the particular mammalianparathyroid hormone which can be measured by an RIA incorporating suchantibodies are designated herein as C-terminal fragments of theparticular mammalian parathyroid hormone.

To prepare Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent, purchase crude bovine PTH TCApowder (Inolex Corp., Glenwood, Ill. 60425) and purify it byconventional means. Dissolve 40 mg of the powder in 10 ml 0.2 M aceticacid-ammonium acetate buffer. Add to the dissolved powder both asuspension of blue dextran and a quantity of Na¹²⁵ I representingapproximately 100,000 cpm. Layer this mixture onto a G-75 Sephadex60×1.5 cm fine column using downward flow and eluate with aceticacid-ammonium acetate buffer. The first peak represents the void volumeas marked by the blue dextran. The second peak represents the includedvolume as marked by ¹²⁵ I. Collect eluates on a 1.25 minute basisbetween the void and included volume and assay each tube to confirm thepresence of purified labeled bPTH. Pool tubes containing immunoreactivebovine PTH and assay pooled material to determine the quantity thereof.

Inject 0.1 mg of the purified labeled bovine PTH emulsified with anequal volume of Freund's complete adjuvant, weekly for four weeks, infour sites near wings and legs on the underside of the boides of RhodeIsland or Leghorn Red chickens. In the fifth week, bleed the chickensfrom a vein under the wing and test for good titer.

Centrifuge the chicken's blood to isolate the plasma. Titer the plasmaagainst ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴. Prepare dilutions of 1/500, 1/1,000,1/5,000, 1/10,000, 1/20,000, and 1/30,000 by volume, by addingappropriate quantities of 1% BSA--0.1 M borate buffer to the isolatedchicken plasma. Add to 200 μl of each dilution of chicken plasma 200 μlof ¹²⁵ I-Tyr⁶⁵⁻⁸⁴ Reagent (cf. preparation of ¹²⁵ I-Tyr⁶⁵⁻⁸⁴ Reagentinfra). Vortex gently and incubate for 24 hours at 2°-8° C. Follow theprotocol (cf. protocol infra) beginning with the addition of RAC-PPTReagent (cf. preparation of RAC-PPT Reagent infra). Plot percent bindingof the chicken antibody to ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ against dilution ofthe chicken plasma according to the counts registered for precipitatedand non-precipitated ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴. Good titer is representedby the dilution which has at least 40-50% binding.

Bleed the animal weekly if good titer is present. Maintain animalthereafter by injecting 0.05 mg of the purified labeled bovine PTHemulsified with an equal volume of Freund's complete adjuvant.

Substantially maximal sensitivity of the assay kit, on the order of 0.2ng/ml hPTH¹⁻⁸⁴ equivalents, is achieved by maintaining a pool of chickenanti-hPTH⁶⁵⁻⁸⁴ plasma solution for which there is approximately 40-50%binding. The pool is maintained by adding to it chicken anti-hPTH⁶⁵⁻⁸⁴plasma at a concentration in 1% BSA--0.1 M borate buffer which accordingto its titer will result in 40-50% binding.

in the case of the presently existing pool, Chicken Anti-hPTH⁶⁵⁻⁸⁴Reagent is prepared by diluting 1.0 ml of the pooled plasma solution to100 ml with 1% BSA--0.1 M borate buffer. One ml of the 1/100 dilution isdiluted to 48 ml with 1% BSA--0.1 M borate buffer. Five ml of the1/4,800 dilution is aliquoted per 30 ml Wheaton vial, lyophilized, andstored at -20° C. Add 25 ml of distilled water to reconsitute for use asChicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent.

PREPARATION OF RABBIT ANTI-CHICKEN PRECIPITATING COMPLEX REAGENT

Rabbit Anti-Chicken Precipitation Complex Reagent (RAC-PPT Reagent) isthe result of raising a rabbit antibody against the gamma globulin ofchickens. This particular rabbit antibody was chosen for its highaffinity for chicken anti-hPTH⁶⁵⁻⁸⁴. The rabbit antibody is added topolyethylene glycol (PEG) and normal chicken serum (NCS) to form apre-precipitated accelerated precipitating complex.

As alternative embodiments, other antibodies with a substantiallysimilar affinity for chicken anti-hPTH⁶⁵⁻⁸⁴ may be utilized in aC-terminal PTH RIA kit. Such antibodies may be raised in animals otherthan rabbits. Also such antibodies may be raised in rabbits and otheranimals against substances other than this particular chicken gammaglobulin, such as whole chicken serum or plasma.

To prepare the RAC-PPT Reagent, separate gamma globulin from the chickenplasma for which there was good titer against ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ bythe conventional method of salting out with 331/3% saturated (NH₄)₂ SO₄.Inject 1.0 mg of the gamma globulin emulsified with an equal volume ofFreund's complete adjuvant, weekly for four weeks, in four sites nearthe legs on the underside of New Zealand White rabbits. In the fifthweek, bleed each rabbit from a vein under a leg and test the titer.

Centrifuge the rabbit's blood to isolate the plasma. To find theconcentration or titer of rabbit antibody, prepare dilutions of 1/2,1/3, 1/4, 1/5, 1/7.5, 1/10, 1/15, 1/20, 1/25, 1/30, and 1/50 by volume,by adding appropriate quantities of 1% BSA--0.10 M borate buffer to theisolated rabbit plasma. To reaction vessels corresponding to eachdilution, add 200 μl of Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent to 200 μl of ¹²⁵I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ Reagent. Vortex gently and incubate 20 hours at 2°-8°C. To each of the reaction vessels, add 200 μl of 1/200 normal chickenserum (NCS does not contain the chicken anti-hPTH⁶⁵⁻⁸⁴ antibody), 200 μlof 15% PEG--0.001% Triton, and 200 μl of one of the dilutions of therabbit plasma. Vortex gently and incubate for exactly two hours at 2°-8°C. Follow the protocol infra from thereon.

Plot the ratio of the cpm of the precipitate to total cpm of precipitateand supernatant against the dilution of rabbit plasma. Maximal bindingof the rabbit antibody to chicken anti-hPTH⁶⁵⁻⁸⁴ occurs at theconcentration of rabbit plasma for which the ratio of cpm's is greatest.This concentration is defined as the working concentration or titer ofthe rabbit antibody.

Bleed the animal weekly if good titer is present. Maintain animalthereafter by injecting 0.5 mg of the chicken gamma globulin emulsifiedwith an equal volume of Freund's complete adjuvant.

To prepare RAC-PPT Reagent, mix equal volumes of NCS at 1/40 dilution in5% BSA--0.25 M borate buffer and rabbit plasma at five times the workingconcentration diluted with 5% BSA--0.25 M borate buffer. Mix well andaliquot 12 ml per 100 ml vial. Add 22.5 ml of 15% PEG--0.001% Triton toeach vial. Lyophilize and store at -20° C. Add 100 ml of distilled waterto reconsitute for use as RAC-PPT Reagent.

PREPARATION OF ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ REAGENT

The ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ Reagent is the result of three principalsteps: (1) synthesizing Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ ; (2) labeling the synthesizedcompound with ¹²⁵ I; and (3) dissolving the labeled compound in anappropriate buffer at an appropriate concentration.

As alternative embodiments, the hPTH⁶⁵⁻⁸⁴ fragment may be labeledradioactively in many conventional ways, including but not limited to,attaching histidyl, tyramyl, histamyl, or other similar substances tothe fragment and labeling with radioiodine. As an alternative to ¹²⁵ I,¹³¹ I may be used. As a further alternative embodiment, a radioactivelylabeled fragment within the range of about 65-84 of bovine PTH or hPTHcould be subsituted for the radioactivley labeled hPTH⁶⁵⁻⁸⁴.

1. Synthesizing Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴

The compound Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ consists of the following: NH₂-Tyr-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asp-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln-COOH.

Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ is synthesized by solid state peptide synthesis, amethod discovered by R. B. Merrifield and detailed in Stewart & Young,Solid State Peptide Synthesis (W. F. Freeman & Co. 1969).

The Synthesis proceeds from the carboxyl end of the hPTH sequence,adding one amino acid per cycle of reaction. In general, each amino acidto be added in protected by BOC (tertiary butyloxycarbonyl) at itscarboxyl end. The peptide chain is linked by its carboxyl end to DVBresin (a copolymer of styrene and divinyl benzene). The startingmaterial, BOC-Gln-DVB resin, is available commercially (PeninsulaLaboratories, Inc., Belmont, Calif. 94002). Place 1.0 mM (i.e. 2.0 gm)BOC-Gln-DVB resin in a reaction vessel. The BOC group is cleaved fromthe Gln-DVB resin by a surplus (hereinafter a quantity sufficient tocover the resin in the reaction vessel) of 40% solution by volume oftrifluoro acetic acid in CH₂ Cl₂. Stir for 20 minutes and drain. Thereaction vessel and contents should be at room temperature for this andall succeeding steps in the synthesis. Then wash with a surplus of pureCH₂ Cl₂, stir and drain. The Gln-DVB resin is neutralized by adding 20ml of 10% solution by volume of triethyamine in CH₂ Cl₂. Stir for 10minutes and drain. Then wash with a surplus of pure CH₂ Cl₂, stir anddrain. Repeat washing twice with fresh CH₂ Cl₂.

In the next cycle, add 5 molar equivalents (defined according to theGln-DVB resin) of the next amino acid in the hPTH⁶⁵⁻⁸⁴ sequence. In thisparticular cycle, the compound added would be BOC-Ser-Bzl (Benzylgroup). Certain amino acids, such as Ser, Lys, Thr, Asp, Glu, and Tyr,require side chain protection to ensure that the amino acid remainsintact throughout the synthesis. This is accomplished by employingBOC-Ser-Bzl rather than BOC-Ser in the initial step of the Ser cycle.Similarly, BOC-Lys-Cl-Z (Chlorocarbobenzosy), BOC-Thr-Bzl, BOC-Glu-Bzl,and BOC-Tyr-Br-Z (Bromocarbobenzosy) are substituted in the reaction forthe unprotected BOC-amino acid. No protection is required for Ala, Leu,Val, and Gly. The above mentioned unprotected and protected BOC-aminoacids are available commercially (Peninsula Laboratories, Inc., Belmont,Calif. 94002). For alternative embodiments, BOC-histamyl, BOC-histidyl,and BOC-tyramyl, along with appropriate side chain protection, areavailable commercially (Peninsula).

To the Gln-DVB resin and the BOC-Ser-Bz, add 5 molar equivalents ofdichlorohexycarbodiamide dissolved in a surplus of CH₂ Cl₂. Stir for 2hours. Apply ninhydrin test. If the test yields negative results, thereaction is complete and the contents of the reaction vessel should bewashed three times with a surplus of CH₂ Cl₂. If the test yields apositive result, the reaction should be continued until complete. Afterwashing, the BOC group is cleaved and the peptide-resin is neutralizedby the steps set forth above. The cycle is repeated with an appropriateprotected or unprotected BOC-amino acid until Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ -DVBresin, or the appropriate alternative embodiment, is the result.

The completed peptide, Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴, is cleaved from the DVB resinand the side protecting groups are stripped off the protected amino acidby washing the contents of the reaction vessel with 20 ml of anhydrousHF. The peptide is purified by countercurrent exchange usingbutanol-acetic acid-water in the ratio of 4:1:5. The purity of thepeptide can be confirmed by thin layer countercurrent chromatography andHPLC.

2. Labeling Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴

The preferred labeling material is ¹²⁵ I, although other materials couldbe used. The labeling procedure stems from Yalow and Berson, 212 Nature357 (1966) and Hunter & Greenwood, 194 Nature 495 (1962). Na¹²⁵ I in astandard NaOH solution is manufactured weekly by Amersham Searle(Arlington Heights, Ill. 60005). Na¹²⁵ I that is older than two weeksshould not be used. Chloramine T is manufactured by Matheson, Colemanand Bell (Cincinnati, Ohio 45212). The stability of Chloramine T isunknown. Chloramine T older than six months should not be used. Sodiummetabisulfite is commonly available.

Sephadex G-100, fine grade 40-20 microparticle diameter, should beutilized in the labeling procedure. A 60×1 cm column at room temperatureshould be packed in a level position. The Sephadex slurry is poured downa glass rod into the column. After the column is poured, add 4 ml of 5%BSA--0.1 M borate buffer and approximately 0.5 ml of 0.5% blue dextran.Run two volumes of 0.1 M borate buffer through the column to pack itthoroughly and to wash excess BSA off the column. The blue dextranchecks column packing and determines void volume. To achieve this, theeluate is collected into a graduated cylinder beginning with theaddition of the blue dextran until the blue dextran comes off thecolumn.

Pick a gamma counter with linear response in the range of 50,000 to200,000 cpm (Tracor Analytic, Elk Grove Village, Ill. 60007). Place atwo millicurie sample of Na¹²⁵ I in the standard NaOH solution in aV-vial. Adjust the gamma counter so that the counts are kept between50,000 and 200,000 cpm by adjusting the distance of the V-vial from thegamma counter, the geometry of the well of the counter, and the gammacounter's window. Add 50 μl of 0.5 M phosphate buffer to the V-vial. Thestrong buffer is necessary to neutralize the NaOH. Cover tightly andvortex until the meniscus disappears. Add 60 μl of 0.1 μg/μl of Tyr⁶⁴-hPTH⁶⁵⁻⁸⁴ in 0.1 N acetic acid by placing the pipet tip containing thatsolution below the meniscus in the V-vial and very gently blowing. Afterten seconds, add 10 μl of Chloramine T solution by similarly gentlyblowing it below the meniscus. Cover tightly and mix. The Chloramine Tsolution should be prepared within 1 hour of its use by diluting 12.5 mgof Chloramine T to a volume of 10.0 ml with 0.05 M phosphate buffer.After mixing the contents of the V-vial for 30 seconds, add 25.0 μl ofsodium metabisulfite solution below the meniscus and vortex. The sodiummetabisulfite solution is made by diluting 12.5 mg of sodiummetabisulfite to a volume of 10.0 ml with 0.05 M phosphate buffer.Transfer the V-vial's contents to the top of the dry Sephadex bed with aPasteur pipet that was rinsed with 5% BSA--0.1 M borate buffer. Thelatter helps prevent labeled Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ from sticking to thepipet. Allow the V-vial's contents to soak into the bed. Rinse vial with200 μl of 5% BSA--0.25 M borate buffer by repeatedly washing down sidesof V-vial with the same BSA. Transfer the 200 μl wash to the column withthe same Pasteur pipet. Allow to soak into bed. Operate the fractioncollector manually during sample and rinse application. Rinse sides ofcolumn with a small amount of 0.1 M borate buffer (usually 0.25-0.5 ml)and allow it to soak into the bed. Fill the area of column above the bedwith 0.1 M borate buffer and attach reservoir. With a fractioncollector, collect 0.5 ml eluates per 1.25 minutes. Collectapproximately 100 fractions.

Count V-vial in which labeling reaction took place and count eacheluate. Plot eluate counts on plain graph paper with counts on theordinate and eluate number on the abscissa. The first peak is generallyconsidered damaged PTH fragment and is discarded. The third peakrepresents free salt. The second peak is the good labeled hormone and issaved and pooled into a plastic bottle using a Pasteur pipet to transferthe solution. Use 20 ml minus the estimated volume of the pooled eluateof 5% BSA--0.25 M borate buffer for rinsing the eluate tubes and thePasteur pipet. Mix the pooled eluates, and any unused 5% BSA--0.25 Mborate buffer.

After plotting the eluate counts against the eluate number, separate theprotein counts (first two peaks) from free salt (the third peak). Thepercent transfer may be calculated by the formula: ##EQU1## The specificactivity may be calculated by the formula: ##EQU2## A specific activitywithin the range of 100-200 μc/μg is acceptable. A count of the reactionvial should not yield a result that is more than 5% the total counts ofthe original vial and contents. If the count exceeds 5%, the number inexcess of 5% should be considered to be protein associated and thereforemust be deducted from the amount of protein applied to the column. Theamount of protein applied to the column may be calculated by theformula: ##EQU3## The amount of labeled protein per container may becalculated by the formula: ##EQU4##

3. ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ Reagent

Dilute the pooled eluate representing the second peak to a sufficientvolume with 5% BSA--0.25 M borate buffer in 0.22 M EDTA so that theconcentration of ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ is 1.25 nanograms/ml based onthe preceding formula for calculating the amount of labeled protein percontainer. Aliquot 5 ml of ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ at a concentration of1.25 nanograms/ml into 30 ml serum vials. Store this solution at -20° C.until it is lyophilized. Store at -20° C. after lyophilization. Thelyophilized ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ stored at -20° C. has a useful lifeof 6 weeks. Reconstitute the lyophilized ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ with 25ml of distilled water as ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ Reagent. Perform a checkon the labeling procedure by directing an assay incorporating the newlyprepared ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ Reagent against known amounts of Tyr⁶⁴-hPTH⁶⁵⁻⁸⁴.

PREPARATION OF CONTROLS AND STANDARDS

Prepare the Normal Control Reagent from plasma units availablecommercially. Centrifuge to remove fibrin or filter through glass wool.Mix pool well and aliquot 2.0 ml into a 5 ml Wheaton vial using asufficiently accurate pipetting device. Assay to determine ng/ml ofhPTH¹⁻⁸⁴ equivalents. Store at -20° C. until lyophilized, and store at-20° C. thereafter. Reconstitute with 2.0 ml of distilled water asNormal Control Reagent.

Prepare the High Control Reagent from plasma units availablecommercially. Centrifuge as indicated above. Add 1000 ng of bovine PTHto 1000 ml of pooled plasma, mix well. Assay to determine ng/ml ofhPTH¹⁻⁸⁴ equivalents. Aliquot 2.0 ml into 5 ml Wheaton vials usingsufficiently accurate pipetting device. Store at -20° C. thereafter.Reconstitute with 2.0 ml of distilled water as High Control Reagent.

Prepare the Zero Standard and the Nonspecific Binding Standard Reagent(NSB Reagent) by placing 8.0 ml of freshly prepared 1% BSA--0.25 Mborate buffer into each of two vials, lyophilize, and store at -20° C.Reconstitute each with 20 ml of distilled water as Zero Standard Reagentand as NSB Reagent respectively.

Prepare Standard Reagents of 0.312, 0.625, 1.25, 2.5, 5.0, and 10.0ng/ml of hPTH¹⁻⁸⁴ equivalents as follows. Dissolve and dilute 0.5 mgTyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ to 5.0 ml with 0.1 N acetic acid. One hundred μl ofthis solution is further diluted to 100 ml with 1% BSA--0.1 M boratebuffer. This results in a 100 ng/ml concentration of Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴.Dilute 1.0 ml of this solution with 39.0 ml of 1% BSA--0.1 M boratebuffer to achieve a working dilution of 2.5 ng/ml which is equal to 10.0ng/ml of hPTH¹⁻⁸⁴ equivalents. Serially dilute the 10.0 ng/ml ofhPTH¹⁻⁸⁴ equivalents standard with 1% BSA--0.1 M borate buffer to give5, 2.50, 1.25, 0.625, and 0.312 ng/ml of hPTH¹⁻⁸⁴ equivalents standards.Aliquot 500 μl of each standard in 2.5 ml serum vials. Dispensing volumeshould remain in range of 500±5.0 μl. Store at -20° C. before and afterlyophilizing. Reconstitute each standard with 500 μl of distilled wateras the respective Standard Reagent.

All of the lyophilized standards have a useful life of at least oneyear.

PROTOCOL

1. Reconstitute lyophilized reagents as indicated.

2. Label 12×75 mm glass tubes in duplicate according to the needs ofprotocol. Commercially available tubes with lowest nonspecific bindingare those of CMB Dispo, SP Dispo, and Kimble (Curtis MathesonScientific, Inc., Houston, Tex. 77001).

3. Add reagents to the tubes as follows:

a. Nonspecific binding (NSB)

250 μl of NSB Reagent

b. Zero standard

50 μl of Zero Standard Reagent.

200 μl of Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent.

c. hPTH standards (0.312 ng/ml--10 ng/ml)

50 μl/tube respectively of each Standard Reagent.

200 μl of Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent.

d. Quality control

50 μl/tube respectively of High Control and Normal Control Reagents.

200 μl of Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent.

e. Unknown sera

50 μl of unknown serum

200 μl of Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent.

4. Vortex gently and incubate 4 hours at 2°-8° C.

Incubation can be extended to 24 hours for increased sensitivity.

5. Add 200 μl of ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴ Reagent to all tubes.

6. Vortex gently and incubate 20 hours at 2°-8° C.

7. Add 500 μl of RAC-PPT Reagent.

8. Vortex gently without foaming and incubate at room temperature for aminimum of 2 hours or at 2°-8° C. for a minimum of 16 hours.

9. Centrifuge the tubes for 20 minutes at a minimum of 760×g.

10. Decant supernatants in separate 12×75 mm tubes.

11. Count the supernatant and its related precipitate, each for asufficient time to achieve statistical significance.

As an alternative embodiment, the above protocol may be altered so thatthe 200 μl of Chicken Anti-hPTH⁶⁵⁻⁸⁴ Reagent and 200 μl of ¹²⁵ I-Tyr⁶⁴-hPTH⁶⁵⁻⁸⁴ Reagent are added as one step to the NSB Reagent, variousstandard reagents, control reagents, and unknown serum. This initialstep may be followed by vortexing gently and incubating for 16 hours at2°-8° C. However, greater assay sensitivity is generally obtained by theorder of reactions indicated by the preferred embodiment.

RESULTS

The concentration of intact hPTH and C-terminal hPTH fragments in theunknown human serum are determined by reference to a standard curve.Standard curves may be obtained by several methods, for example, byplotting percent binding vs. concentration or by plotting cpm ofprecipitate, or the supernatant, or the ratio therebetween vs.concentration. FIG. 3 shows a sample standard curve where percentbinding is plotted vs. concentration in ng/ml of hPTH¹⁻⁸⁴ equivalents.The percent binding may be calculated by the formula: ##EQU5##

As an alternative embodiment, the results of the nonspecific binding(NSB) sample may be incorporated in the calculation of the percentbinding. NSB, not to be confused with NSI, is the result of radioactivesupernatant residing in the precipitate and protein binding to the wallsof the assay tubes, as well as other factors. An alternative calculationshould be used when the NSB sample results in precipitate cpm'sindicating greater than 5% binding. The % binding for a sample may becalculated in the alternative by the formula: ##EQU6## The alternativecalculation of % binding is used for % binding of NSB samples thatexceed 5%. For binding of NSB samples of 5% or less, the distortion dueto NSB is thought to be substantially uniform for all samples. The RIA'sbased on the foregoing protocol typically result in 5% binding for NSBsamples.

As another alternative embodiment, the assay may be run with 100 or 200μl of standards and unknown serum for greater sensitivity at lowconcentrations. FIG. 4 compares standard curves for 50, 100, and 200 μlof standard and unknown serum. Conversely, if the concentration of anunknown serum sample exceeds 10 ng/ml, a second assay of the serum pooldiluted by 0.4% BSA--0.1 M borate buffer will yield the concentrationafter applying an appropriate dilution factor.

Data for the purposes of illustration are shown below. These numberswere used to plot the standard curve in FIG. 3 which results in aconcentration of 3.5 ng/ml of hPTH¹⁻⁸⁴ equivalents for the unknown serumsample.

    ______________________________________                                                                                Calcu-                                                  Average        Known  lated                                         Average   (2 tubes)      ng/ml  ng/ml                                         (2 tubes) cpm of         hPTH.sup.1-84                                                                        hPTH.sup.1-84                                 cpm of    precip-  %     equival-                                                                             equival-                              Sample  supernatant                                                                             itate    binding                                                                             ents   ents                                  ______________________________________                                        NSB     14,265      735     4.9% --     --                                    Standard                                                                              8,100     6,900    46.0% 0      --                                            8,175     6,825    45.5% 0.312  --                                            8,550     6,450    43.0% 0.625  --                                            9,000     6,000    40.0% 1.25   --                                            9,945     5,055    33.7% 2.5    --                                            11,250    3,750    25.0% 5.0    --                                            13,125    1,875    12.5% 10.0   --                                    Quality                                                                       Control -                                                                     Normal  8,445     6,555    43.7% 0.5    0.5                                   High    9,555     5,445    36.3% 2.0    2.0                                   Unknown 10,500    4,500    30.0% --     3.5                                   ______________________________________                                    

I claim:
 1. A compound having the formula X⁶⁴ -hPTH⁶⁵⁻⁸⁴ wherein X is amember of the group consisting of histidyl, tyramyl, histamyl, andtyrosyl.
 2. The compound of claim 1 wherein said member is tyrosyl.
 3. Acompound having the formula Y-X⁶⁴ -hPTH⁶⁵⁻⁸⁴ wherein Y is a member ofthe group consisting of ¹³¹ I and ¹²⁵ I and X is a member of the groupconsisting of histidyl, tyramyl, histamyl, and tyrosyl.
 4. The compoundof claim 3 wherein said compound is ¹²⁵ I-Tyr⁶⁴ -hPTH⁶⁵⁻⁸⁴.
 5. Acompound having the formula X⁶⁴ -bovine PTH⁶⁵⁻⁸⁴ wherein X is a memberof the group consisting of histidyl, tyramyl, histamyl, and tyrosyl. 6.The compound of claim 5 wherein said member is tyrosyl.
 7. A compoundhaving the formula Y-X⁶⁴ -bovine PTH⁶⁵⁻⁸⁴ wherein Y is a member of thegroup consisting of ¹³¹ I and ¹²⁵ I and X is a member of the groupconsisting of histidyl, tyramyl, histamyl, and tyrosyl.
 8. The compoundof claim 7 wherein said compound is ¹²⁵ I-Tyr⁶⁴ -bovine PTH⁶⁵⁻⁸⁴.